Mario is invited to Peach's castle, but when he arrives Peach is nowhere to be seen. He soon learns from Toad that Bowser has once again kidnapped her. The Koopa King has also put a spell on her castle, imprisoning her subjects. So it is up to Mario to break the spell and rescue Peach.

Super Mario 64 is the first Mario game done entirely with 3D graphics. The core gameplay is similar to previous platform installments, focusing on jumping, avoiding obstacles, and defeating enemies. Levels can be explored without time limits. Stars must be collected in order to unlock new areas, eventually gaining access to various parts of Bowser's castle. Defeating Bowser on each floor procures keys necessary to unlock big doors and enter other floors, which contain more areas.

Mario has more moves at his disposal in this installment. Besides running and jumping, he can now walk, crouch, crawl, climb, and punch enemies. Double and triple jumps, long jumps, wall jumps, and backflips can be executed as well. Swimming underwater now depletes Mario's oxygen level. Items must be picked up and carried in some instances in order to solve puzzles.

Power-ups include the Wing Cap, which allows Mario to fly; the Metal Cap, which protects him from damage (including environmental hazards such as poisonous gas); and the Vanish Cap, which makes Mario ethereal, allowing him to walk through certain obstacles (such as wire mesh). Mario can also enter cannons and be shot from them; combining this with the flying ability grants access to high areas.